This invention relates to protein or proteinaceous protease inhibitors and to novel variants thereof, useful in conjunction with enzymes in cleaning compositions. The invention provides inhibitor variants greater proteolytic stability in detergent. The present invention also relates a variety of cleaning compositions comprising these inhibitors, and the genes encoding them.
Enzymes make up the largest class of naturally occurring proteins. Cleaning compositions may include many different enzymes to accomplish stain removal. For example, a liquid laundry detergent may contain proteases, lipases, amylases, peroxidases, and cellulases. The protease""s ability to hydrolyze proteins has been exploited in cleaning compositions by incorporating proteases as an additive to aid in removing peptide or protein stains.
However, a commonly encountered problem in such protease-containing liquid aqueous detergents is the degradation by protease of the protease itself or of other enzymes (such as, lipase, amylase and cellulase) in the composition during storage. As a result of this degradation, the detergent composition consequently performs less well. Therefore it is commercially useful to incorporate into the cleaning composition a protease inhibitor.
There is a need to provide inhibitors that are stable enough to be useful. This xe2x80x9cusefulnessxe2x80x9d is measured in terms of the need to provide a long shelf life for the cleaning composition, and an improved yield of the protease from the biological host.
Additionally, these inhibitors could be useful for cleaning compositions, regardless of the composition type, e.g., liquid, gel, granular, or solid compositions.
It would be advantageous to provide reversible inhibitors of the protease, so that upon dilution of the composition during cleaning, or in the cleaning environment, the protease is no longer inhibited, but rather is able to hydrolyze the peptide stains.
Synthetic Protease Inhibitors
Various synthetic protease inhibitors or stabilizers have been proposed for such uses. Panandiker et al. (U.S. Pat. No. 5,422,030) discloses an aromatic borate ester to stabilize enzymes in laundry detergents. For instance, U.S. Pat. No. 4,566,985 proposes to use benzamidine hydrochloride, EP 376 705 proposes to use lower aliphatic alcohols or carboxylic acids, EP 381 262 proposes to use a mixture of a polyol and a boron compound. Such synthetic approaches to enzyme inhibition may provide longer shelf life, but may be expensive and may not improve isolation yield due to proteolysis in the fermentor.
Recognizing these shortcomings, those in the art have experimented with proteinaceous protease inhibitors in hopes of stabilizing enzymes in cleaning compositions, without the drawbacks of the synthetic inhibitors.
Proteinaceous Protease Inhibitors
Nature provides proteinaceous protease inhibitors to regulate the protease in its natural environment (i.e., in vivo). However, these proteinaceous protease inhibitors tend to be unstable, therefore, their commercial use in the presence of proteases and detergents may be somewhat limited.
Proteinaceous protease inhibitors are typically long peptides (often over 28 amino acids), which bind to the active site of a protease and inhibit its activity. These inhibitors have been classified into several families (Families I to IX) based on primary amino acid sequence homologies (Laskowski, M., Jr., and I. Kato, xe2x80x9cProtein Inhibitors of Proteinasesxe2x80x9d, Ann. Rev. Biochemistry, (1980) 49: 593-626). Included in these inhibitors are those commonly referred to as family VI inhibitors, such inhibitors include eglin and barley chymotrypsin inhibitor, and family III inhibitors, such as Streptomyces subtilisin inhibitor (SSI), and plasminostreptin.
Such inhibitors tend to bind to specific proteases better than others. Thus it is convenient to consider the inhibitor with a specific protease in mind. For this reason, the art often discusses them as xe2x80x9cprotease/peptide inhibitor pairs.xe2x80x9d An example of a known protease/peptide inhibitor pair is subtilisin BPNxe2x80x2/SSI. See for example, Y. Mitsui, Y. et al, xe2x80x9cCrystal Structure of a Bacterial Protein Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) at 2.6 A Resolutionxe2x80x9d, J. Mol. Biol. 131: 697-724, (1979) and S. Hirono, H. Akagawa, Y. Mitsui, and Y. Iitaka, xe2x80x9cCrystal Structure at 1.6 A Resolution of the Complex of Subtilisin BPNxe2x80x2 with Streptomyces Subtilisin Inhibitorxe2x80x9d, J. Mol. Biol. 178: 389-413, (1984). Mikkelsen (published application WO 92/03529) discloses peptide inhibitors of family VI. It is said that these inhibitors stabilize lipase and cellulase to proteolysis. Mikkelsen recognizes that many natural inhibitors have a high affinity for the protease and that the inhibitor-enzyme complex does not dissociate upon dilution into the wash environment. Mikkelsen discloses the use of proline in the Family VI P1 position. It is recognized that if the protease is completely inhibited in the product, then only a small fraction of the protease would be active even after dilution in the cleaning environment.
SSI is stable in the presence of subtilisin BPNxe2x80x2, as long as the inhibitor is present in sufficient amounts to inhibit all protease activity. However, SSI is unstable in the presence of excess protease.
Tamura et al, (Biochemistry 30: 5275-5286, 1991) suggests that SSI""s instability in the presence of excess protease is due to dissociation and conformational change of hydrophobically formed SSI dimers. Tamura discloses a D83C variant of SSI displaying a higher Tm than native SSI using DSC (Tamura et al., Biochemistry 33:14512-14520, 1994), but Tamura apparently did not test protease resistance in the presence or absence of detergent.
However, if the binding constant (Ki) of an inhibitor provides for some protease activity in the cleaning composition containing the enzyme/inhibitor pair, the protein inhibitor, as well as enzymes in the composition, may be hydrolyzed. Therefore, it would be advantageous to find variants of SSI or other inhibitors which are suitably stable in the presence of protease as well as detergents. In addition, it is preferred that these inhibitors have a binding constant for the particular protease to be inhibited. This binding constant (Ki) should allow for inhibition of the protease in the cleaning composition and during its storage. However, upon diluting the cleaning composition or during the cleaning process, the protease and inhibitor dissociate, and the uninhibited protease becomes active.
The binding of some protease inhibitors has been investigated. Halkier et al. (WO 93/20175) discloses protease inhibitors (e.g., eglin and barley chymotrypsin inhibitors) with lowered affinity compared to naturally occurring family VI inhibitors, due to changes at the P1 and P4 to P2 positions of the inhibitor.
Since the amino acid sequence of any protein or peptide determines its characteristics, a change in the amino acid sequence may alter the protein""s or peptide""s properties depending upon the location and nature of the amino acid change. Thus mutagenesis has been employed on some protein protease inhibitors in an attempt to determine the structure or function of certain amino acids therein.
For example, Kojima et al. (S. Kojima, Y. Nishiyama, I. Kumagai, and K. Miura, J. Biochem. 109: 377-382, 1991) made and measured the Ki of 19 SSI P1 position variants against wild-type SSI using subtilisin BPNxe2x80x2. As another example, Mikkelsen discloses mutations in family VI inhibitors that are said to lower binding affinity. Nielsen et al. (WO 93/17086) discloses changes to plasminostreptin that are said to lower binding affinity.
The art describes the need for proteinaceous protease inhibitors that are useful. For example, Feder and Kochavi (FR208475 1) disclose a reversible alkaline protease inhibitor which is said to stabilize an enzyme in the presence of detergents. Estell (U.S. Pat. No. 5,178,789) discloses the use of turkey ovomucoid as a reversible inhibitor said to be useful for stabilizing subtilisin.
Of course, for cost reasons and the like, it would be advantageous to provide inhibitors useful at very low levels in cleaning compositions. In addition to this advantage, such an inhibitor would allow for the use of enzymes which are highly sensitive to proteolytic degradation in compositions comprising a protease.
Despite the work in this area, it remains that protein protease inhibitors are generally too unstable in the presence of protease and detergents to be commercially useful.
Despite the variety of approaches described in the art, there is a continuing need for new, and more effective variants of proteases useful for cleaning compositions.
For example, if used in a laundry application, it would be desirable that any inhibitor dissociate from the protease upon dilution in the washing machine, enabling the protease to be active. Because the dilution in a washing machine is finite, the art recognizes that the inhibitor need not render the protease completely inactive in the product, but should render the protease active in the cleaning environment. It would be advantageous to have about 0.01% to about 1% of the protease be free from inhibitor in a composition. If the inhibitor is used in a liquid cleaning composition, those in the art desire inhibitors which are stable in the presence of free protease.
It is an object of the present invention to provide peptide or proteinaceous protease inhibitor variants having greater proteolytic stability, especially in detergent, and lower affinity for the protease than the wild-type inhibitor.
It is also an object of the present invention to provide cleaning compositions comprising these variants.
The present invention provides peptide or proteinaceous protease inhibitor variants having a modified amino acid sequence from wild-type amino acid inhibitors; wherein the modification positions provides greater proteolytic stability and lower affinity for the protease. The present invention also provides the genes encoding such variants.
The present invention also provides compositions comprising such variants for cleaning.
Other benefits that the invention provides will be apparent as the description of the invention proceeds.
We have found variants of the protease inhibitors which are more stable to free (i.e., uninhibited) protease in vitro and in the presence of surfactants, including detergents. In addition, it is contemplated that such variants are more stable in vivo as well.
We have also found protease inhibitors with suitable inhibition constants (Ki), that provide for suitable inhibition of protease during growth, harvesting, purification and in the cleaning composition. This provides for better stability and longer shelf life (i.e., storage time that the product remains substantially unchanged in its properties.) It is contemplated that better stability results in decreased autolysis and thus increases yield of protease from the organism.
The preferred compositions of the invention comprise three essential ingredients: (A) protein protease inhibitor(s) or a mixture thereof, (B) protease(s) or a mixture thereof, and (C) a surfactant. The compositions according to the present invention may further comprise optional ingredients, including other enzymes.
The term xe2x80x9cprotease inhibitorxe2x80x9d, as used herein, means any reversible inhibitor of the proteolytic activity of the protease enzyme. This inhibitor is a protein or polypeptide. Typically, the inhibitor is long enough to be called a protein in its own right, this means that the polypeptide is at least 25 amino acids in length, more preferably at least 28 amino acids, most preferably at least 80 amino acids in length.
Preferably the inhibitor moiety is resistant to proteolysis by the corresponding protease.
This invention pertains to peptide protease inhibitor variants, that have been modified by mutating the various nucleotide sequences that code for the inhibitor, thereby modifying the amino acid sequence of the inhibitor. The modified peptide protease inhibitor variants (hereinafter, xe2x80x9cvariantsxe2x80x9d) of the present invention have improved stability to proteases, and inhibit protease in the formulation, but dissociate upon dilution (i.e., they xe2x80x9cdilute offxe2x80x9d) in the cleaning environment.
The present invention also pertains to the genes encoding for such variants.
Preferred embodiments of this invention are useful for many proteases for which SSI is an inhibitor. These include, but are not limited to, Savinase, subtilisin BPNxe2x80x2, subtilisin Carlsberg, and their derivatives.
Without being bound to any theory, this dissociation upon dilution may be improved by modifying the amino acid sequence to provide variants which have protease binding constants (Ki) which differ from the wild-type proteinaceous protease inhibitors. This improved Ki allows for dissociation in the cleaning process or upon dilution.
The total protease is the sum of inhibitor-bound protease, substrate-bound protease, and free protease. When an inhibitor has the optimal Ki, it provides nearly complete inhibition at the concentration typical in the stored cleaning composition (i.e., on the shelf) and nearly complete dissociation (i.e., lack of inhibition) at the concentration typical in use (e.g., in the washer, in a solution for soaking, etc.). Thus the protease/inhibitor must be diluted to provide active protease.
Preferred variants of the invention are designed to alter the inhibition of the proteases to provide these desirable properties. Preferably this alteration allows the inhibitor to inhibit the protease in the stored product and dissociate upon dilution. These binding affinities, or inhibition constants (Ki) are tailored using site-directed mutagenesis. Furthermore, the affinity of the inhibitor for the protease is optimized for each different protease, and each cleaning application.
In terms of a Ki, a xe2x80x9csuitable Kixe2x80x9d, xe2x80x9cadvantageous Ki, desirable Ki, or optimal Kixe2x80x9d, as referred to herein, allows the inhibitor to inhibit nearly all protease (preferably above 60%, more preferably about 99%) in the cleaning composition or product, but still allows the inhibitor to dissociate from the protease upon dilution.
For example, where a 1:1 stoichiometry is used, it is preferred that inhibitors have a Ki value between 10xe2x88x9210 and 10xe2x88x924, depending on the application. As another example, a liquid laundry composition, as commonly used in the U.S., preferably has an inhibitor with a Ki between 10xe2x88x9210 and 10xe2x88x926, assuming the composition is diluted 600-fold in the washing machine. Of course, should washing machine dimensions or product concentrations change, the Ki is adjusted accordingly. Prediction of a useful Ki range is readily determined by the skilled artisan without undue experimentation by considering such parameters as dilution of the cleaning formula upon use, temperature dependence of the binding constant in relation to the temperature of cleaning method used, stoichiometry of the inhibitor to the protease, and the like.
In a preferred embodiment, the preferred stoichiometry is 1:1 to about 3:1 (inhibitor:protease), preferably 1.5:1 to about 3:1 (inhibitor:protease), more preferably about 2:1 (inhibitor:protease). In this context, the xe2x80x9csuitable Kixe2x80x9d as referred to herein, allows the inhibitor to inhibit nearly all protease (preferably about 99%) in the cleaning composition or product, but still allows the inhibitor to dissociate from the protease upon dilution. Because of the change in stoichiometry, however, it is preferred that inhibitors have a Ki value between 10xe2x88x9210 and 10xe2x88x924, depending on the application. As an example, a liquid laundry composition, used in the U.S., preferably has an inhibitor with a Ki between 10xe2x88x9210 and 10xe2x88x926, assuming the composition is diluted 600-fold in the washing machine, as above. Of course, should washing machine dimensions, product concentrations, or temperature of the wash solution change, the Ki is adjusted accordingly.
Thus the amount of inhibitor used in a cleaning composition is defined by the amount of the protease to be inhibited in the cleaning composition.
As used herein, xe2x80x9cvariantxe2x80x9d means an inhibitor having an amino acid sequence which differs from that of the known wild-type, this variation may be by substitution of one or more amino acids, or by deletion or addition of amino acids either at the ends or in the sequence of the variant. Thus the term xe2x80x9cvariantxe2x80x9d includes xe2x80x9cinhibitor likexe2x80x9d peptides that have additional amino acids, even if the variant sequence contains the entire wild type sequence.
As used herein, xe2x80x9cgenexe2x80x9d, xe2x80x9cvectorxe2x80x9d, xe2x80x9cplasmidxe2x80x9d, xe2x80x9cgenomexe2x80x9d, or xe2x80x9cchromosomexe2x80x9d have their art recognized meanings. However, the skilled artisan will recognize that teaching how to use an expression system using a plasmid, etc. is sufficient to teach the skilled artisan how to use other systems whether they be genomic, plasmid-based, etc., whether they are used in procaryotes or eucaryotes, and whether the heterologous host is bacterial, fungus, plant, etc.
Since the peptide is ultimately encoded in vivo by DNA, the DNA can be used to define the inhibitor sequences. The DNA, which codes for the inhibitor or its variants, can be used in any number of plasmids and/or expression systems, including in vitro expression systems and in vivo systems such as plants, (preferably those used in biotechnology, including tobacco, oilseed plants, such as rapeseed, soybean and the like, grain, such as maize, barley, oats, other vegetables, such as tomatoes, potatoes and the like) and microorganisms, including fungi, such as yeast, and bacteria, such as Bacillus, E. coli and the like. Preferably the expression system is a microorganism, more preferably bacterial in nature, most preferably E. coli or Bacillus, still more preferably Bacillus.
It is understood that the skilled artisan, given the instruction of this application, will appreciate that the DNA used to code for the inhibitor or its variants, may be placed in the same plasmid, phage or chromosome as other inhibitors of the invention. In addition, such plasmids, phages or chromosomes may also encode proteases, including fusion proteins which include as part of the fusion protein an inhibitor and/or protease, which may or may not be inhibited by the inhibitor of the invention.
The DNA encoding the inhibitor or its variant may be incorporated into a plasmid, or phage, active in the cell, or may be incorporated directly into the genome of the organism which is used in cloning or expression of the inhibitor of the invention.
It is also well understood by the skilled artisan that the DNA described above also contemplates, and discloses the RNA transcript of the DNA. The skilled artisan can, without experimentation, know the RNA sequence, by inspection of the DNA sequence.
As used herein, xe2x80x9cmutant genexe2x80x9d means a gene coding for a variant.
As used herein, xe2x80x9cwild-type inhibitorxe2x80x9d refers to known and naturally derived protease inhibitors. An example of one of these inhibitors is Streptomyces Subtilisin Inhibitor (SSI). SSI is 113 amino acids in length and is described by Obata et al. (xe2x80x9cMolecular Cloning and Nucleotide Sequence Determination of Gene Encoding Streptomyces Subtilisin Inhibitor (SSI)xe2x80x9d, S. Obata, S. Taguchi, I. Kumagai, and K. Miura, J. Biochem. 105: 367-371 (1989), incorporated herein by reference. SEQ ID NO:19 shows the wild-type SSI gene of Obata et al. As used herein, the amino acid numbering is that of Obata et al. As used herein, this amino acid numbering is applied to other subtilisin inhibitors from Streptomyces, in accordance with Terabe et al. (infra).
We use a synthetic SSI gene, designed to be rich in adenine and thymine, as is B. subtilis DNA. The sequence of the synthetic SSI gene is represented by SEQ ID NO:1. This synthetic gene encodes two extra amino acid residues at the peptide""s amino terminus due to expression plasmid construction methods. This modified amino acid sequence, including four additional amino acids, is represented by SEQ ID NO:2. As used herein, the term xe2x80x9cwild-type amino acid sequencexe2x80x9d and xe2x80x9cwild-type inhibitorxe2x80x9d is represented by SEQ ID NO:2.
SSI may exist in dimeric form. Thus without being bound by theory, it is possible that stabilizing dimeric SSI provides increased protease resistance to excess protease. Preferably this stabilized dimeric SSI variant is composed of two SSI variant monomers covalently bound together. This may be by ester, amido, disulfide, or other linkages, commonly occurring in amino acids and their sidechains. Thus xe2x80x9ccovalent dimerizationxe2x80x9d and xe2x80x9ccovalent stabilizationxe2x80x9d refer to such covalently bound monomers, which form the dimer. Preferably this dimerization occurs via disulfide bonds.
For example, we have found that a cysteine at amino acid residue 83 stabilizes SSI. It might also stabilize related inhibitors, such as plasminostreptin, SIL1, SIL2, SIL3, SIL4, STI2, API-2cxe2x80x2, SLP1, SIL10, SIL13, SIL14 (xe2x80x9cThree Novel Subtilisin-Trypsin Inhibitors from Streptomyces: Primary Structures and Inhibitory Propertiesxe2x80x9d, M. Terabe, S. Kojima, S. Taguchi, H. Momose, and K. Miura, J. Biochem. 116: 1156-1163, 1994) and other homologous inhibitors from Streptomyces.
A second essential ingredient in the present detergent compositions is from about 0.0001% to 1.0%, preferably about 0.0005% to 0.2%, most preferably about 0.002% to 0.1%, weight % of active protease. Mixtures of protease are also included. The protease can be of animal, plant or, preferably, microorganism origin. Preferred for use herein are subtilisin-type proteases. Particularly preferred is bacterial serine protease (or a variant thereof) obtained from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus lentus, and/or Bacillus licheniformis. 
Of course, the weight percent of protease in the cleaning composition will vary depending on the water content, builder content and the like of the finished composition. For example, it is preferred that in a granular detergent, 0.064 to about 0.64 mg/g of protease in the composition is desirable. The skilled artisan will appreciate that a gel detergent resembles a liquid formulation. Thus weight percent of protease may be adjusted to compensate for the variables above detergent (as well as others understood in the art).
Suitable proteases include Novo Industries A/S AlcalaseR (preferred), EsperaseR, SavinaseR (Copenhagen, Denmark), Gist-brocades"" MaxataseR, MaxacalR and Maxapem 15R (protein engineered MaxacalR) (Delft, Netherlands), and subtilisin BPN and BPNxe2x80x2(preferred), which are commercially available. Preferred proteases are also modified bacterial serine proteases, such as those made by Genencor International, Inc. (San Francisco, Calif.) which are described in European Patent Application Serial Number 87303761.8, filed Apr. 28, 1987 (particularly pages 17, 24 and 98), and which is called herein xe2x80x9cProtease Bxe2x80x9d, and 199,404, Venegas, published Oct. 29, 1986, which refers to a modified bacterial serine protease (Genencor International) which is called xe2x80x9cProtease Axe2x80x9d herein (same as BPNxe2x80x2). Preferred proteases, then, are selected from the group consisting of AlcalaseR (Novo Industries A/S), BPNxe2x80x2, Protease A and Protease B (Genencor), and mixtures thereof. Protease B is most preferred.
From about 1 to 80, preferably about 5 to 50, most preferably about 10 to 30, weight % of surfactant is the third essential ingredient in the present invention. The surfactant can be selected from the group consisting of anionics, nonionics, cationics, ampholytics, zwitterionics, and mixtures thereof. Although the compositions according to the present invention are preferably used in the context of laundry cleaning, said compositions according to the present invention can be used in other different cleaning applications including hard surface cleaning, or dishwashing. The particular surfactants used can therefore vary widely depending upon the particular end-use envisioned.
The benefits of the present invention are especially pronounced in compositions containing ingredients that are harsh to enzymes such as certain detergency builders and surfactants.